Stable liquid gonadotropin formulation

ABSTRACT

The present invention pertains in general to the field of the stabilization of gonadotropin formulations, in particular liquid formulations of gonadotropins. The stabilization is achieved by a particular combination of excipients, preferably arginine and methionine. In a preferred embodiment, the formulation does not comprise a buffer.

FIELD OF THE INVENTION

The present invention pertains in general to the field of thestabilization of gonadotropin formulations, in particular liquidformulations of gonadotropins. The stabilization is achieved by aparticular combination of excipients, preferably arginine andmethionine. In a preferred embodiment, the formulation does not comprisea buffer.

BACKGROUND

Gonadotropins are a family of hormones, which are essentially and mainlyinvolved in the fertility cycle in females and males. Gonadotropins canbe derived from urine, both for research and treatment purposes,however, several gonadotropins like e.g., hCG, LH and FSH, can also beproduced recombinantly.

In particular, gonadotropins can be employed in the treatment ofinfertility.

The four main gonadotropins all belong to the same glycoprotein family.These are follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH) and (human) chorionicgonadotropin (hCG). All of these gonadotropins are heterodimeric andconsist of an alpha and a beta subunit; the alpha subunit is common toall, i.e. the same for all above-mentioned four gonadotropins, while thebeta subunit differs, respectively. The action of FSH is mediated by adistinct FSH receptor. The beta chains of LH and HCG share 82% proteinsequence homology and exert their actions through the same LH receptor.

FSH is naturally secreted by the anterior pituitary gland and functionsto support follicular development and ovulation. FSH comprises a 92amino acid alpha subunit, also common to the other glycoproteinhormones, e.g. LH and hCG, and a 111 amino acid beta subunit unique toFSH that confers the biological specificity of the hormone (Pierce andParsons, 1981, Glycoprotein hormones: structure and function, Ann RevBiochem., 50: 465-495). The mature beta subunit of hCG is composed of145 amino acids. Each subunit in FSH and hCG is post-translationallymodified by the addition of complex carbohydrate residues. For FSH, bothsubunits carry two sites for N-linked glycan attachment, the alphasubunit at amino acids 52 and 78 and the beta subunit at amino acidresidues 7 and 24 (Rathnam and Saxena, (1975) Primary amino acidsequence of follicle stimulating hormone from human pituitary glands. I.alpha subunit, J Biol Chem. 250 (17):6735-6746; Saxena and Rathnam,(1976) Amino acid sequence of the beta subunit of follicle-stimulatinghormone from human pituitary glands, J Biol Chem. 251(4): 993-1005)).FSH is thus glycosylated to about 30% by mass (Dias and Van Roey, (2001)Structural biology of human follitropin and its receptor. Arch Med Res.32(6): 510-519; Fox et al. (2001) Three-dimensional structure of humanfollicle-stimulating hormone. Mol Endocrinol. 15(3), 379-89). The betasubunit of hCG contains both N- and O-glycosylation (N-13, N-30, O-121,O-127, O-132 and O-138). The extra glycosylation in the beta subunit ofhCG makes it more hydrophilic than that of FSH. R-subunits providespecificity for the receptor interaction.

Urinary derived gonadotropins have been used clinically for over 40years and their safety is well established. New generations of highlypurified (HP) urinary derived gonadotropin compared with the firstgeneration have been introduced over time. The increased purity isobtained by adding additional purification steps, such as anion exchangeand hydrophobic interaction chromatography steps to remove urinaryproteins without FSH and/or LH bioactivity. The significantly increasedpurity of the new generation gonadotropin preparations facilitates morecomprehensive characterization studies providing additional informationon the composition.

Purified urinary FSH and human menopausal menotropins (hMG), bothisolated from the urine of post-menopausal women, have been used formany years in infertility treatment either to induce (mono) ovulation orto stimulate multiple follicles in patients undergoing controlledovarian stimulation (COS) prior to assisted reproduction technologies(ART). Two recombinant versions of FSH, Gonal-F® (follitropin alpha,Merck Serono) and Puregon®/Follistim® (follitropin beta, Merck) becameavailable in the mid-1990s. Both products are expressed in Chinesehamster ovary (CHO) cell lines (Howles, C. M. (1996), geneticengineering of human FSH (Gonal-f), Hum Reprod. Update, 2:172-191).

CHO cells are commonly used for the production of pharmaceuticalrecombinant proteins. Structural analysis has identified that sialicacid is exclusively attached by an α2,3-linkage. Many humanglycoproteins contain a mixture of both α2,3- and α2,6-linkages forsialic acid residues. Therefore, recombinant proteins expressed usingthe CHO system will differ from their natural counterparts in their typeof terminal sialic acid linkages.

Infertility

In the present context, “infertility” shall be defined as the diminishedability or the inability to conceive and have offspring. Women who areable to get pregnant but then have repeat miscarriages are also said tobe infertile. Infertility is also defined in specific terms as thefailure to conceive after a year of regular intercourse withoutcontraception. Infertility can be due to many causes. Studies have shownthat a little more than half of cases of infertility are a result offemale conditions. The remaining cases are caused by sperm disorders andby unexplained factors. There are currently several possibilities totreat infertility.

Those possibilities are a timed intercourse, the use of assistedreproductive technologies (ARTs), a medical management of endometriosis,ovulation induction (OI), fibroids and female sexual dysfunction (FSD),and surgery to correct abnormalities.

In assisted reproductive technology and OI, drugs to stimulate ovulationare used. Next to FSH, that is primarily responsible for the ovarianstimulation, gonadotropin preparations may contain LH and/or hCG.

Several different drug products containing gonadotropin hormones derivedfrom urine of pregnant or postmenopausal women are currently used inclinical practice for the treatment of infertility, such as HMG (humanmenopausal gonadotropin) preparations containing a 1:1 ratio of FSH andLH bioactivity (see e.g. USP version 35, monograph for menotropins), aswell as preparations containing only FSH bioactivity. From 1995 onwards,gonadotropin products, manufactured by recombinant DNA technology, havebecome available.

It is therefore important to provide stabilized formulations of suchgonadotropin compounds, either alone or in a mixture.

Thus, it is an aim of the present invention to provide formulations, inparticular liquid formulations, of one or more gonadotropins,particularly of a composition comprising hCG, optionally in acombination with FSH, which are stable. It is a further object of thepresent invention to provide a method for stabilization of suchformulations. It is another object to provide such a formulation whichis stable for 12 months, preferably for 24 months, even more preferablyfor 24 months at storage conditions plus 1 month “in use”, i.e. at roomtemperature.

SUMMARY OF THE INVENTION

The present invention pertains to stable liquid gonadotropinformulations. In a preferred embodiment these formulations compriseshCG. In an also preferred embodiment, this formulation comprises bothFSH and hCG. The gonadotropins in the formulations of the invention arepreferably urinary-derived or plasma-derived, but can in an alternativeembodiment be recombinantly produced.

In the following, the term “hMG” shall be used interchangeably with“gonadotropins from urine”. Mostly, the gonadotropins from urine will befrom human urine.

Human chorionic gonadotropin (hCG) contributes LH (luteinizing hormone)activity, which is responsible for presently approved pharmaceuticalindications. This is a well known fact, and is described as part of theSmPCs of hMG preparations like the Menopur®-product, authorized for thesame indications as presently claimed. Menopur®—in these SmPCs—comprisesFSH and LH activity, but additionally confirms that hCG is responsiblefor at least part of the LH activity. Thus, any reference to hCG in thecontext of the present invention encompasses formulations which comprisean LH activity which is attributable to hCG.

Preferred embodiments include the following:

-   -   1. A liquid pharmaceutical gonadotropin formulation, comprising        a gonadotropin, arginine in an amount of from 50 to 160 mM, and        methionine in an amount of from 0.05 to 1.5 mg/ml, wherein the        formulation does not comprise an additional buffer, and wherein        the pH of the formulation is between 6.0 and 7.5.    -   2. The pharmaceutical formulation of item 1, wherein the        gonadotropin comprises hCG (human chorionic gonadotropin), and        optionally FSH and/or LH.    -   3. The pharmaceutical formulation of item 1 or 2, wherein the        gonadotropin comprises hMG (human menopausal gonadotropin).    -   4. The pharmaceutical formulation of any one of items 1 to 3,        wherein the gonadotropin (such as FSH, LH and/or hCG) is of        human origin and urinary-derived.    -   5. The pharmaceutical formulation of any one of items 1 to 3,        wherein the gonadotropin (such as FSH, LH and/or hCG) is        recombinant.    -   6. The pharmaceutical formulation of any one of the preceding        items, additionally comprising a preservative, preferably        phenol.    -   7. The pharmaceutical formulation of any one of the preceding        items, additionally comprising a surface-active agent,        preferably a polysorbate, even more preferably polysorbate 20.    -   8. The pharmaceutical formulation of item 6 or 7, wherein the        preservative, preferably the phenol, is present in an amount of        4-6 mg/ml, preferably in an amount of 5 mg/ml.    -   9. The pharmaceutical formulation of item 7 or 8, wherein the        surface-active agent, preferably the polysorbate 20, is present        in an amount of 0.001-0.05 mg/ml, preferably in an amount of        0.005 mg/ml.    -   10. The pharmaceutical formulation of any one of the preceding        items, wherein the arginine is preferably L-arginine HCl.    -   11. The pharmaceutical formulation of any one of the preceding        items, wherein the hMG is present in an amount of 300-900, more        preferred 500-700, even more preferred 600-650, very preferably        625 IU/ml.    -   12. The pharmaceutical formulation of any one of the preceding        items, which consists of        -   625 IU/ml hMG        -   0.15 mg/mi Methionine        -   150 mM arginine        -   5 mg/ml phenol        -   0.005 mg/ml polysorbate 20        -   Water for injection (WFI), and        -   wherein the formulation has a pH of 6.8+/−0.3.    -   13. A liquid pharmaceutical formulation as described in any one        of the preceding items, for use in a method of treatment of        infertility.    -   14. The pharmaceutical formulation for the use of item 13,        wherein the treatment is a treatment of ovulation induction        (OI), assisted reproductive techniques (ART), and/or        hypogonadotrophic hypogonadism in men.    -   15. A method for stabilization of a liquid pharmaceutical        formulation, comprising hMG, which comprises the step of        -   Providing a sample of urine from a human woman        -   Extracting hMG        -   Compounding said extract with arginine and methionine, in            amounts as defined in any of the preceding items,        -   Optionally further adding phenol and polysorbate in amounts            as defined in any of the preceding items,        -   Adjusting the pH to provide a formulation with a pH between            6.0 and 7.5,        -   wherein no additional buffer is added.

As described above, gonadotropins, e.g. FSH and hCG as well as LH aresuitable for the treatment of infertility. In that regard, it has becomeclear that liquid formulations of these gonadotropins can be unstable;this is true even for those gonadotropins which are destined for singleuse. Instability can be even more pronounced if the liquid formulationscomprise a preservative, which is e.g. prescribed for all multidoseformulations.

The present formulations may be destined for single-use or formulti-use, respectively.

The FSH formulated in the present formulations may be is urinary orplasma-derived or recombinant FSH (rFSH). In a preferred embodiment, theFSH is urinary or rFSH; particularly preferred it is urinary FSH.

As mentioned above, it is now possible to produce gonadotropins, likeFSH, LH or hCG recombinantly. Thus, reference here to a gonadotropin ingeneral always includes both the urinary- or plasma-derived derived aswell as the recombinant (r) gonadotropin, unless otherwise specified.Thus, e.g. reference to “FSH” also encompasses rFSH. The production andamino acid sequences as well as the nucleic acid sequences of FSH, hCGand LH are all well-known to the person skilled in the art.

The sequences which can be used in the context of the present inventionare as follows:

FSH, LH and hCG alpha subunit(see also Fiddes, J.C. and Goodman, H.M. The gene encoding the common alpha subunitof the four human glycoprotein hormones J. Mol. Appl. Genet. 1(1), 3-18(1981))(SEQ ID NO: 1)MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS (underlined part is the leader peptide) (116)FSH beta subunit(see also Saxena, B.B. and Rathnam, P. Amino acid sequence of the beta subunit offollicle-stimulating hormone from human pituitary glands J. Biol. Chem. 251(4), 993-1005(1976))(SEQ ID NO: 2)MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPSYCSFGEMKE (underlined part is the leaderpeptide) (129) hCG beta subunit(see also Fiddes, JC, Goodman HM. The cDNA for the beta-subunit of human chorionic gonadotropinsuggests evolution of a gene by readthrough into the 3′-untranslated region Nature. 1980 Aug. 14;286(5774): 684-7) (SEQ ID NO: 3)MEMFQGLLLLLLLSMGGTWASKEPLRPRCR PINATLAVEK EGCPVCITVN TTICAGYCPT MTRVLQGVLPALPQVVCNYR DVRFESIRLP GCPRGVNPVV SYAVALSCQC ALCRRSTTDC GGPKDHPLTC DDPRFQDSSSSKAPPPSLPS PSRLPGPSDT PILPQ (underlined part is the leader peptide) (165)LH beta subunit(see also Sairam, M.R. and Li, C.H. Human pituitary lutropin. Isolation, properties, and thecomplete amino acid sequence of the beta-subunit Biochim. Biophys. Acta 412(1), 70-81(1975))(SEQ ID NO: 4) MEMLQGLLLLLLLSMGGAWASREPLRPWCH PINAILAVEKEGCPVCITVNTTICAGYCPTMMRVLQAVLPPLPQVVCTYRDVRFESIRLPGCPRGVDPVVSFPVALSCRCGPCRRSTSDCGGPKDHPLTC DHPQLSGLLFL (underlined part is the leader peptide) (141)

In an alternative embodiment, the rFSH or the rHCG of all embodiments isa long-acting rFSH or rhCG, respectively. Such e.g. long-acting FSHformulations can be obtained as generally known to a person skilled inthe art, e.g. by modifying the FSH molecule or by modifying theformulation.

“FSH” as used herein thus encompasses all possible urinary derived orrecombinant forms of the above-mentioned FSH as well as all possiblecombinations of FSH forms. Also encompassed is a formulation for singleuse and one or more further formulations (of the same or a differentgonadotropin) for multi-dose use.

One possible product may be a formulation including FSH (in a preferredembodiment also including hCG, and/or optionally LH, LH activity etc.),all in different containers. The LH activity, if present, may originatefrom LH or hCG. LH can be replaced by an equivalent dose of hCG and viceversa; an “equivalent dose” in that context can be calculated as is wellknown in the art.

A particularly preferred gonadotropin combination is that of FSH andhCG, preferably as an hMG formulation in one container, but optionallyalso e.g. in different containers, like e.g. vials or cartridges.

Possible combinations, which can be provided also in differentcontainers, also include: urinary (u)FSH and uhCG or uFSH and uLH;further (rhCG or rLH or rFSH) and (uhCG or uLH or rhCG or rLH), and allpossible permutations thereof. In a very preferred embodiment, theinventive formulation comprises FSH and hCG. In another equallypreferred embodiment, the inventive formulation comprises hCG.

The gonadotropin formulations of the present invention are liquidformulations. Preferably, the formulation is injectable. Formulationscan be supplied as a product having one, two or more pharmaceuticalcomposition(s) including FSH or FSH/hCG, and/or LH, for administrationseparately or together. If administered separately, administration canbe sequential. The product can be supplied in any appropriate package.For example, a product can contain a number of pre-filled syringes eachincluding FSH (an FSH composition), or additionally hCG (an hCGcomposition) e.g. wherein the syringes can be packaged in a blisterpackage or other means to maintain sterility. A product can optionallycontain instructions for using the gonadotropin formulations.

According to a further aspect, the inventive gonadotropin formulation isprovided as a multi-dose preparation. The present invention, however,explicitly is also directed to formulations destined for a single use.The present invention also pertains to a stabilization of formulationsas part of a kit. Such a kit will comprise at least one containercomprising one or more daily doses of the gonadotropin, e.g. FSH, ore.g. two containers (e.g. a vial), each comprising a differentgonadotropin like hCG, and e.g. further instructions (e.g. foradministration) and e.g. further means for injection. In a preferredembodiment, an injection pen for multiple injections is used, wherebythe gonadotropin solution is filled in respective cartridges. The activeingredients can be in different cartridges, but can of course beinjected simultaneously, or in sequential order, as is well known to theperson skilled in the art. Also, two or more active ingredients can bewithin one and the same cartridge.

In a very preferred embodiment, the present formulation is forparenteral use, even more preferred for subcutaneous injection.

In a preferred embodiment, the hMG is present in the formulation in anamount of 35-850 IU/ml, preferably 50-800 IU/ml, even more preferred100-700 IU/ml, most preferred 625 IU/ml, typically in a multidoseformulation.

A particularly preferred formulation of excipients for a multidoseformulation comprising hMG as above, and/or comprising hMG, and/or hCG,and/or all other gonadotropins as mentioned as coming under the presentinvention, has the following composition:

-   -   50-160 mM arginine HCl, preferably 150 mM, arginine HCl    -   0.05 to 1.5 mg/ml, preferably 0.15 mg/ml, L-methionine    -   0.001-0.05 mg/ml, preferably 0.005 mg/ml, Polysorbate 20    -   4.0 to 6.0 mg/ml, preferably 5.0 mg/ml, phenol    -   pH 6.0 to 7.5, preferably pH 6.8+/−0.3, (the pH refers to the pH        of the whole solution.)

WFI.

Typical concentrations of the active ingredient for formulationscomprising recombinant hCG and/or FSH are as follows, although theconcentration of the active ingredient does not have any influence onthe performance of the present invention:

For rFSH: 30-150 μg/ml

For rhCG: 5-200 μg/ml

The preferred excipients for such recombinant formulations are the sameas described above for the multidose hMG formulation. Typicalsingle-dose formulations are also encompassed in this invention andwould be the same as described above with the exception that they wouldnot comprise a preservative, like phenol.

Injectable depot forms can be made by forming micro encapsulatedmatrices of the gonadotropin (and other agents, if present) inbiodegradable polymers. The polymer based depot forms/sustained releasesystems can, dependent on their chemical nature, be for example micro-or nanoparticles, hydrogels, micelles, emulsions or implants. Dependingupon the ratio of gonadotropin to polymer and the nature of theparticular polymer employed, the rate of gonadotropin release can becontrolled. Examples of biodegradable polymers includepolylactide/polyglycolide copolymer systems, polyvinylpyrrolidone,poly(orthoesters), poly(anhydrides), poly(ethylene glycol), poly aminoacids, polysaccharides e.g. sodium hyaluronate (NaHA) or other saltshereof, gelatin, chitosan etc. All mentioned polymers can be derivatizedor modified to optimize the protein drug delivery or its stability.Depot injectable formulations are also prepared by entrapping thegonadotropin in lipid systems, or polymer lipid mixtures as micelles,liposomes or micro-emulsions which are compatible with body tissues.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents. It is possible to form sterile solid compositionswhich can be dissolved or dispersed in sterile water or other sterileinjectable medium just prior to use.

Injectable formulations can be supplied in any suitable container, e.g.vial, pre-filled syringe, injection cartridges, and the like, asdescribed above.

The pH and exact concentration of the various components of aformulation for use as a pharmaceutical composition as described hereinare principally adjusted in accordance with routine practice in thisfield, see e.g. The textbook of Pharmaceutical Medicine, fifth edition,edited by John P. Griffin and John O'Grady. In a preferred embodiment,the compositions of the invention are supplied as compositions forparenteral administration. General methods for the preparation of theparenteral formulations are known in the art and are described inREMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY, supra, at pages780-820. The parenteral compositions can be supplied in liquidformulation or as a solid which will be mixed with a sterile injectablemedium just prior to administration. In an especially preferredembodiment, the parenteral compositions are supplied in dosage unit formfor ease of administration and uniformity of dosage.

The FSH, hCG and/or LH that can be formulated in accordance with thepresent invention can be derived by conventional means from urine, as iswell known in the art, or can be produced recombinantly. For possibleproduction methods it is further referred to e.g. WO 2009/127826.

One preferred embodiment of the invention is the presently describedformulation comprising hCG.

hCG can be obtained by any means known in the art. hCG as used hereinincludes urinary-derived hCG and recombinant hCG. A formulationcomprising urinary derived hCG is particularly preferred. A formulationhaving LH activity which is—at least partly—derived from hCG (in otherwords: the hCG is the molecule which is responsible for this LHactivity) is also encompassed. Human-derived hCG can be purified fromany appropriate source (e.g. urine and/or placenta) by any method knownin the art. Methods of expressing and purifying recombinant hCG are wellknown in the art.

LH can be obtained by any means known in the art. LH, as used herein,includes human-derived LH and recombinant LH. Human-derived LH can bepurified from any appropriate source (e.g. urine) by any method known inthe art. Methods of expressing and purifying recombinant LH are known inthe art.

The term “pharmaceutical composition” is used herein interchangeablywith “pharmaceutical formulation”.

The stable pharmaceutical composition of the present invention may beused for the treatment of infertility. “Treatment of infertility” in thecontext of this invention includes treatment of infertility bycontrolled ovarian (hyper)stimulation (COS) or methods which include astep or stage of controlled ovarian stimulation, for example IntraUterine Insemination (IUI), in vitro fertilisation (IVF), orintracytoplasmic sperm injection (ICSI). The term also includesovulation induction (OI) or methods which include a step or stage ofovulation induction. The term also includes treatment of infertility ina subject having tubal or unexplained infertility, including treatmentof infertility in a subject having endometriosis, for example stage I orstage II endometriosis (as defined by the American Society forReproductive Medicine (ASRM) classification system for the variousstages of endometriosis, Revised American Society for ReproductiveMedicine dassification of endometriosis: 1996, Fertil Steril 1997; 67,817-821), and/or in a subject with a partner with male factorinfertility. The term preferably includes the use in e.g. assistedreproductive technologies (ARTs), ovulation induction (OI) orintrauterine insemination (IUI). The pharmaceutical composition may beused, for example, in medical indications where known FSH preparationsor preparations of FSH and hCG are used. In a typical embodiment, thepresent formulation is used for the same medical indications as thoseindications approved for Menopur®, in Europe, as follows, in anexemplary case:

Treatment of Female and Male Infertility:

-   -   Anovulatory women: MENOPUR can be used to stimulate follicle        development in amenorrhoeic patients. Clomiphene (or a similar        ovulation inducing agent which influences steroid feed-back        mechanisms) is the preferred treatment for women with a variety        of menstrual cycle disturbances, including luteal phase        insufficiency with anovulatory cycles and with normal prolactin,        and also amenorrhoeic patients with evidence of endogenous        oestrogen production but normal prolactin and normal        gonadotropin levels. Non-responders may then be selected for        menotrophin therapy.    -   Women undergoing superovulation within a medically assisted        fertilisation programme: MENOPUR can be used to induce multiple        follicular development in patients undergoing an assisted        conception technique such as in-vitro fertilisation (IVF).    -   Hypogonadotrophic hypogonadism in men: MENOPUR may be given in        combination with human chorionic gonadotropin (e.g. Choragon)        for the stimulation of spermatogenesis.

or the indications as approved for Menopur® in the US as follows:

Development of multiple follicles and pregnancy in ovulatory women aspart of an Assisted Reproductive Technology (ART) cycle.

Alternatively, the treatment options with Menopur® can be described asfollows:

MENOPUR is indicated for the treatment of infertility in the followingclinical situations: Anovulation, including polycystic ovarian disease(PCOD), in women who have been unresponsive to treatment with clomiphenecitrate.

Controlled ovarian hyperstimulation to induce the development ofmultiple follicles for assisted reproductive technologies (ART) (e.g. invitro fertilisation/embryo transfer (IVF/ET), gamete intra-fallopiantransfer (GIFT) and intracytoplasmic sperm injection (ICSI)).

Stimulation of follicular development in women with hypogonadotrophichypogonadism.

The present invention also provides the use of the stabilizedgonadotropin formulations described herein (according to aspects of theinvention) for, or in the manufacture of a medicament for, the treatmentof infertility.

In a preferred embodiment the present inventive formulation is used forovulation induction, assisted reproductive techniques (ART) and/or forhypogonadotrophic hypogonadism in men.

The pharmaceutical compositions can be further formulated intowell-known compositions for any route of drug administration, e.g. oral,rectal, parenteral, transdermal (e.g. patch technology), intravenous,intramuscular, subcutaneous, intracisternal, intravaginal,intraperitoneal, local (powders, ointments or drops) or as a buccal ornasal spray. A typical composition comprises a pharmaceuticallyacceptable carrier, such as aqueous solution, non-toxic excipients,including salts and preservatives, buffers and the like, as described inRemington's Pharmaceutical Sciences fifteenth edition (Matt PublishingCompany, 1975), at pages 1405 to 1412 and 1461 to 87; and USP-NF, TheNational Formulary XIV fourteenth edition (American PharmaceuticalAssociation, 1975), among others. The present invention is directed insome embodiments to the specific provision of a formulation whichexhibits both surprisingly high stability and further advantages, likeno unacceptable coloring, no unacceptable turbidity, reduced or no painupon injection, and reduced or no skin irritation upon injection. Thishas become possible only with the findings of the present inventionwhich shows that particular components enable the advantageousperformance and high stability of the liquid gonadotropin formulationsdescribed herein.

Although gonadotropin formulations have been on the market for severaldecades now, the formulation scientist is well aware that theformulation of these proteins is associated with a number ofdifficulties. These difficulties exist and vary severely on the basis onmany factors, like

-   -   The fact that a protein is being formulated (proteins as such        are difficult to formulate in any case)    -   The fact that the protein is specifically glycosylated        (glycosylation might be influenced by the specific selection of        the manufacturing and the excipients)    -   The specific protein(s) formulated (formulation chemistry varies        significantly depending on the actual protein: it has e.g. been        shown that even closely related proteins like FSH and hCG behave        differently in the same formulation, see e.g. WO 2012/013742)    -   Whether or not the protein was obtained from nature, e.g. urine,        or produced recombinantly    -   Whether a preservative is needed (some preservatives fulfill        their intended goal of protection against microbial growth, but        affect the stability of the final formulation negatively. This        is true for all of m-cresol, phenol and benzyl alcohol (with or        without benzalkonium chloride), which are currently the only        preservatives authorized for use with gonadotropins; in        addition, preservatives can have a negative effect on coloring,        depending on the further excipients and active ingredient        comprised in the formulation)    -   The specific surface-active agent used (these might in some        instances lead to turbidity, again depending on the further        excipients as comprised in a respective formulation)    -   The specific buffer used (e.g. citrate buffer will frequently        lead to pain and skin irritation upon injection)    -   The specific excipients used for stabilization, which will        stabilize different compositions in unpredictable ways.    -   Depending on the excipient used for stabilization which will        stabilize different compositions in unpredictable ways.

Thus, the formulation scientist is faced with a set of multiple problemson the one hand and a set of a multitude of possible excipients on theother hand, leading to a complex problem for providing a formulationwith a good stability on the one hand and no coloring or turbidity orpain upon injection on the other hand.

It was quite surprising, therefore, that the present combination ofarginine and methionine, without the addition of buffer solved theseparticular problems, in the present pH range.

In this regard, “without (the addition of/an additional) buffer” and“does not comprise a buffer” is used synonymously in this application.This expression shall mean that no additional compound is added/presentin the formulation that would be considered to have buffer capacity. Asolution is said to be buffered if it resists changes in the activity ofan ion on the addition of substances that are expected to change theactivity of that ion. Buffers are substances or combinations ofsubstances that impart this resistance to a solution. Buffered solutionsare systems in which the ion is in equilibrium with substances capableof removing or releasing the ion. It refers to the amount of materialthat may be added to a solution without causing a significant change inion activity. It is defined as the ratio of acid or base added (ingram-equivalents/L) to the change in pH units. The capacity of abuffered solution is adjusted to the conditions of use, usually byadjustment of the concentrations of buffer substances (USP NF). Buffercapacity is expressed commonly as the number of equivalents (Eq) ofstrong acid (e.g. HCl) or base (e.g. NaOH) that causes one liter of thesolution in question to undergo one unit change in pH (preferably at oneatmosphere and 21° C.) (Skoog West and Holler, Fundamentals ofAnalytical Chemistry, fifth edition). One equivalent HCl is equal to onemole HCl and one equivalent NaOH is equal to one mole NaOH. In thepresent invention the buffer capacity will be expressed as number ofequivalents (Eq) of strong acid (e.g. HCl) or base (e.g. NaOH) thatcauses one liter of the solution in question to undergo one unit changein pH. Thus, in accordance with the present invention, the formulationshall not comprise an additional buffer, that contributes with ≥0.5mEq/(liter*pH)(preferably at 1 atm, 21° C.) in the pH range as disclosedfor the present inventive formulations.

Method to Determine and Calculate Buffer Capacity

Buffer capacity can be determined and calculated as follows.

A definite volume of the solution to be tested is titrated with acide.g. HCl or base e.g. NaOH.

Suitable concentrations of acid and base e.g. 0.2 N should be used tomake a sufficiently precise titration.

Titration is performed by adding small aliquots of HCl or NaOH to thetest solution. For each addition the added volume and the correspondingpH is documented.

The accumulated volume of acid and base is plotted against the measuredpH, see e.g. FIG. 1 .

A linear least square regression fit is performed for the relevant pHarea and R² is calculated to confirm the validity of the fitted line.

The buffer capacity expressed in mEq/(litre×pH unit) is calculated bylinear regression.

$\begin{matrix}{Y = {\left. {{ax} + b}\Leftrightarrow x \right. = \frac{Y - b}{a}}} & {{Equation}1}\end{matrix}$

where;

x=Buffer capacity [μL 0.2 N HCl/NaOH to move pH 1 pH unit]

Y-b=1 pH unit

a=Slope

and

$\begin{matrix}{x = \frac{\left( {Y - b} \right) \times C}{a \times V \times 10^{6}}} & {{Equation}2}\end{matrix}$

Where mEq is milliequivalents of acid or base is given from theconcentration e.g. 0.2 N HCl/NaOH which is equal to 0.2 equivalents/LHCl/NaOH which is equal to 200 mEq/L HCl/NaOH.

x=Buffer capacity [mEq/litre×pH unit]

Y-b=1 pH unit

C=Concentration of acid or base e.g. for HCl/NaOH

$\left\lbrack {\frac{mmol}{L} = \frac{mEq}{L}} \right\rbrack$

V=Volume of solution in question

10⁶ correction factor from μL to litre

Example of calculation using Equation 2 and batchC-01_(pH range 6.564-6.947), from table 9:

$x = {\frac{1 \times 200}{0.001507 \times 0.1 \times 1000000} = {1.32{mEq}/\left( {{litre} \times {pH}{unit}} \right)}}$

To determine if a component will contribute with 20.5 mEq/(litrexpHunit) to the buffer capacity, the buffer capacity is determined asdescribed above for a solution with the component and for the samesolution without the component. The difference in the determined buffercapacity for the two solutions gives the contribution of the givencomponent to the buffer capacity.

The terms “without (the addition of/an additional) buffer” and “does notcomprise a buffer” also means that the present inventive formulationdoes not comprise any one of the following buffers, (which are FDAapproved buffers for parenteral use in the range of pH 6.0-7.5):

Histidine

Phosphate

Citrate

Tromethamine (Tris)

Hydroxyethylpiperazine Ethane Sulfonic Acid (HEPES)

Carbonate

Additionally, the terms also mean that none of the further FDA approvedbuffers for parenteral use is included, in particular

Acetate

Adipic acid

Ammonium sulphate

Succinate

Asparagine

Aspartic acid

Glutamate (Glutamic acid)

Glycine

Lactate

Lysine

Maleate (Maleic acid)

Fumarate (Fumaric acid)

Malate

Meglumine

Propionate

Alanine

Phenylalanine

Cysteine

Isoleucine

Leucine

Proline

Serine

Tartrate

Threonine

Tryptophan

Tyrosine

Valine

Without the addition of a buffer it is usually assumed that the pH ofthe final solution cannot be kept easily in the desired range (and itwill be difficult to reach a specific target pH during adjustment) (inthe present invention, the pH should be between 6.0 and 7.5, morepreferred between 6.5 and 7.4, preferably at or around 6.8), but willfluctuate severely.

It is extremely important to maintain the correct pH for apharmaceutical product. The pH defines parameters like stability,activity and shelf life. For that reason, pharmaceutical formulationsare usually formulated with a buffer. A variety of buffering agents isavailable and they are selected to be effective at the desired pH.Exemplary buffers which are used regularly in practically all existinggonadotropin formulations are phosphate buffer, and citrate buffer. Thebuffer needs to provide the formulation with a maintained pH over therange of conditions to which the formulation might be exposed duringformulation and in particular during storage thereof. It is often quitechallenging to find such a suitable buffer, and then in some cases aneffective buffer provides the formulation with undesired side-effects,like pain upon injection for citrate buffers. All buffers have theinherent disadvantage of being an additional ingredient in theformulation which complicates the formulation process, poses a risk ofdeleteriously affecting other ingredients, stability, shelf-life, andacceptability to the end user.

The present inventors, however, surprisingly have provided formulationswhere no buffer is present, while the formulations do not suffer fromany unacceptable pH fluctuation. The formulations comprise arginine as astabilizing compound. Arginine would have an expected buffering capacityof +/−1 pH units around its pKa, which is at 2.17, at 12.5 and at 9.04.Thus, no buffering capacity was expected at the preferred pH range ofthis invention (e.g., between 6.0 and 7.5, more preferred between 6.5and 7.4, preferably at or around 6.8). Nevertheless, it was surprisinglydiscovered that arginine has a strong stabilizing influence on the pH inthe particular formulations described herein. No such effect of argininehas been described anywhere in the prior art.

Advantageously, the provision of this formulation without a buffer asdefined herein also provides a formulation with reduced or no pain uponinjection and reduced or no skin irritation upon injection.

This is contrary to formulations which e.g. use citrate buffer which hasbeen shown to lead to skin irritation and pain upon injection.

The arginine of this invention can be arginine, or can be arginine HCl,or even more preferred, L-arginine (HCl). The amount of arginine ispreferably in the range between 50 and 160 mM, more preferred 80-160 mM,even more preferred at approx. 150 mM.

As can be seen from the examples below, arginine has a pronounced effecton oxidation of FSH and hCG. However, the inventors did show aparticularly high stabilizing effect. Quite surprisingly, they were thenable to provide a liquid gonadotropin formulation which was stable andshowed only minimal oxidation (in acceptable levels, see example sectionbelow), if the gonadotropin was combined with a high stabilizing amountof arginine (e.g. 150 mM).

This was particularly surprising as the effect was obtained in a pHrange far removed from the pKa values of arginine, namely in a pH rangeof pH 6.0 to 7.5. This is the preferred pH range for the presentinvention. In a particularly preferred embodiment, the pH is in therange of 6.5 to 7.4. Even more preferred is a pH range between 6.5 and7.2. Most preferred is a pH of 6.8+/−0.3.

Surprisingly, a low anti-oxidant amount of methionine, preferablyL-methionine, was able to prevent undesirable oxidation events. This lowamount could be as low as 0.05 mg/ml, but could go up to 2.5 mg/ml. In apreferred embodiment, the amount of methionine is between 0.1 and 1.5mg/ml. In an even more preferred embodiment the amount of methionine isbetween 0.1 and 1 mg/ml. In an even more preferred embodiment, theamount of methionine is between 0.1 and 0.5 mg/ml. The most preferredembodiment is methionine at 0.15 mg/ml. It could not have been predictedat all that the high oxidation power of arginine could have beencountered and overcome by such a low amount of methionine.

Even further surprising was the finding that arginine had a much morepronounced stabilizing effect than other known stabilizing agents, e.g.sucrose, which is a stabilizer quite frequently added and very wellestablished.

The formulations may include suitable aqueous and non-aqueouspharmaceutical carriers, diluents, solvents and/or vehicles. Examples ofsuitable aqueous and non-aqueous pharmaceutical carriers, diluents,solvents or vehicles include water, ethanol, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like),carboxy-methylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil), and injectable organic esters such as ethyl oleate.

Further preferred, especially for multidose formulations, in the presentinvention is the addition of a preservative. Very preferred thispreservative is phenol. In a further preferred embodiment, the phenol isadded at a concentration of 4.0 to 6.0 mg/ml, preferably 5.0 mg/mlphenol. Surprisingly, the phenol is also advantageous in the context ofthe specific present formulation, compared to other well knownpreservatives, in that it does not lead to a coloring and is verystable, even over a prolonged time period, in spite of the presences ofthis preservative, as shown in the example section.

Further preferred in the present invention is the addition of a surfaceactive agent. Very preferred this surfactant is a polysorbate, even morepreferred polysorbate 20. In a further preferred embodiment, thepolysorbate 20 is added at a concentration of 0.001-0.05 mg/ml,preferably 0.005 mg/ml Polysorbate 20. Advantageously, the formulationof this invention, compounded with polysorbate 20, does not lead toturbidity and is very stable, even over a prolonged time period, inspite of the presence of this preservative, as shown in the examplesection.

The compositions can also contain further additives in addition to thosealready listed above, such as but not limited to (further)preservatives, wetting agents, emulsifying agents, and dispersingagents. Antibacterial and antifungal agents can be included to preventgrowth of microbes and includes, for example, parabens, chlorobutanol,phenols, sorbic acid, and the like. Furthermore, it may be desirable toinclude tonicity agents. The present inventive formulation however doesnot comprise an additional buffer.

In some cases, to effect prolonged action, it is desirable to slow theabsorption of the gonadotrophin(s) such as FSH (and other activeingredients, if present) from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption then depends upon the rate of dissolution which, in turn,can depend upon crystal size and crystalline form. Alternatively,delayed absorption of a parenterally administered FSH combination formis accomplished by dissolving or suspending the FSH combination in anoil vehicle.

According to the present invention, an effort was made by the inventorsto investigate the effect of certain compounds on the stability of aliquid gonadotropin formulation; here, stabilizing as well asdestabilizing effects of several compounds were investigated.Additionally, the inventors sought to improve the resultant formulationas to its clarity, degree of coloration, and pain upon injection.

The term “stability” can refer to chemical stability, involving covalentmodification in the amino acid sequence, but in the context of proteinstability it can also refer to physical stability, which involveschanges of the protein folded state (i.e. the native state) notincluding covalent bond cleavage.

In the present invention the term “stability” refers to thebio-stability of formulations of gonadotropins, in particular FSH andhCG. Physical instability of a protein formulation may be caused byaggregation of the protein molecules to form higher order aggregates, bydissociation of the heterodimers into monomers, or by any otherconformational change that reduces at least one biological activity ofe.g., FSH proteins (and other active ingredients, if present) includedin the present invention.

A “stable” solution or formulation is one wherein the degree of,aggregation, dissociation, conformational modification, loss ofbiological activity and the like, of proteins therein is acceptablycontrolled, and does not increase unacceptably with time. Stability maybe assessed by methods well-known in the art, including measurement of asample's light scattering, visual inspection of clarity and/orcoloration, absorbance, or optical density, molecular sizedeterminations (e.g. by size exclusion chromatography or field flowfractionation), in vitro or in vivo biological activity and/or bydifferential scanning calorimetry (DSC).

The present formulation is stable for 12 months at storage conditions,preferably 16 months at storage conditions, even more preferred for 24months, even more preferably for 24 months at storage conditions plus 1month “in use”, i.e. at room temperature. Stability at “storageconditions” refers to storage of the formulation in a cooledenvironment, e.g. at 5° C.±3° C. “Room temperature” in the presentcontext is meant to refer to at or below 30° C., preferably 15-25° C.,preferably 18-25° C., most preferred 25±2° C.

To determine stability in the context of the present formulation wellknown (immuno)assays for FSH and hCG were used, which are well known tothe person skilled in the art and described herein consequently only ingeneral terms as follows:

RP-HPLC for Oxidation

Oxidization of proteins was determined by RP-HPLC, by gradient elutionon a C4 column and UV detection at 210 nm.

Enzyme-Linked Fluorescent Assay (ELFA)

The immunoactivity of FSH and hCG was determined in a sandwichimmunoassay performed using an automatic analyser VIDAS® instrument(BioMerieux, France) with VIDAS® kits corresponding to FSH and hCG,respectively.

Size Exclusion Chromatoeraohv (SEC)

Purity of rFSH was determined with size exclusion chromatography (SEC)on an SEC column with protein range 3-70 kDa and UV detection at 210 nm.

Hydrophobic Interaction Chromatography (HIC)

Purity of rhCG was determined with hydrophobic interactionchromatography (HIC) by gradient elution on a column with alkyl amidegroups and UV detection at 220 nm.

FSH and LH Bioassay

FSH and LH potency were determined using biological in vivo assays;Steelman-Pohley (FSH) and seminal vesicle weight gain assay (LH),respectively.

Other methods for assessing stability are well known in the art and canalso be used according to the present invention.

The FSH and LH activity assays are standardized using the FourthInternational Standard for Urinary FSH and Urinary LH, November 2000, bythe Expert Committee on Biological Standardization of the World HealthOrganization (WHO ECBS) and are well known to the person skilled in theart.

It was known that several preservatives have a pronounced destabilizingeffect on gonadotropin formulations and it was found surprisingly herethat the described inventive formulation, in particular comprisingarginine and methionine, but not buffer, was particularly stable—evenover a prolonged time period and even in the presence of phenol.

The presently claimed combination of arginine and methionine has astabilizing effect on a liquid hMG formulation which is in anadvantageous and surprising manner even more pronounced than thestabilizing effects of known stabilizers, like e.g. sucrose. Theimproved stabilization effect compared to the known stabilizers likesucrose is particularly surprising. Further, quite unexpected, thestabilizing effects of the inventive combination was pronounced in spiteof the absence of a buffer.

It has been known from the prior art that there is degradation of FSHoccurring in pharmaceutical FSH formulations and this has been confirmedby examples provided in WO 2012/013742. FSH will degrade both as afunction of time as well as a function of temperature.

It appears that the conformational unfolding of tertiary and secondaryFSH structures occurring upon heating is a two-state transition (whenprotein aggregation is limited). This unfolding may be independent ofsubunit dissociation (changes in the quaternary structure).

Further, it became clear in the prior art that FSH, containing apreservative like benzyl alcohol or phenol, where such preservatives arenecessary, for example as antimicrobial agents in liquid FSHformulations, clearly affect the stability of FSH multidose formulationsin a negative manner. Here, the long term stability of FSH is decreased,the denaturation temperature of FSH is lower, and the already denaturedforms have a lower level of secondary structures than FSH formulationsnot containing preservatives.

The present invention also pertains to a method for stabilization of aliquid gonadotropin formulation wherein the method comprises the step ofan addition of arginine and methionine to said formulation, which doesnot comprise a buffer.

LIST OF FIGURES

FIG. 1 : Titration curve. The slope of the curves shows the buffercapacity, e.g. a steep slope depicts a low buffer capacity.

FIG. 2 : Response optimizer based on 25° C. results. Vertical lines showthe level of L-arginine and L-methionine and the responses in the FSHand hCG immunoassay as Y (in IU/ml). R² _(HCG)92.15% and R² _(FSH):67.31%.

All studies were confirmed by the additionally conducted real-time data.

EXAMPLES Example 1 1 Background and Introduction

Several different drug products containing gonadotropin hormones derivedfrom urine of postmenopausal or pregnant women are used in the treatmentof infertility, such as hMG (human menopausal gonadotropin)preparations. hMG possess Follicle Stimulating Hormone (FSH) andLuteinizing Hormone (LH) activity in a one to one ratio.

FSH, LH and human chorionic gonadotropin (hCG) belong to thegonadotropins family of complex glycoprotein hormones. They areheterodimers composed of an α- and a β-subunit. The 92 amino acidα-subunit is common for these three gonadotropins, the β-subunits areunique, giving them their different biological characteristics(Wolfenson C. et al. 2005, Batch-to-batch consistency of human-derivedgonadotropin preparations compared with recombinant preparations.Reproductive BioMedicine. Vol 10 No. 4:442-454; Shen, S. T., Cheng, Y.5., Shen, T. Y., and Yu, J. Y. 2006, Molecular cloning offollicle-stimulating hormone (FSH)-beta subunit cDNA from duckpituitary. Gen. Comp Endocrinol. 148:388-394; Fox, K. M., Dias, J. A.,and Van, R. P. 2001, Three-dimensional structure of humanfollicle-stimulating hormone. Mol. Endocrinol. 15:378-389; Burova, T.,Lecompte, F., Galet, C., Monsallier, F., Delpech, S., Haertle, T., andCombarnous, Y. 2001, Conformational stability and in vitro bioactivityof porcine luteinizing hormone. Mol. Cell Endocrinol. 176:129-134. Theglycoprotein hormones all lose their bioactivity upon dissociation ofthe non-covalently linked subunits (Alevizaki, M. and Huhtaniemi, I.2002, Structure-function relationships of glycoprotein hormones; lessonsfrom mutations and polymorphisms of the thyrotrophin and gonadotropinsubunit genes. Hormones. (Athens.) 1:224-232).

LH and hCG binds to the same receptor and therefore both possess LHactivity. In hMG the LH activity is originating mostly from hCG.

It is the goal of the present invention to develop a gonadotropinformulation as a liquid formulation, for subcutaneous injection. Formultidose injection, addition of a preservative typically is necessary(Meyer, B. K., Ni, A., Hu, B., and Shi, L 2007, Antimicrobialpreservative use in parenteral products: past and present. J. Pharm.Sci. 96:3155-3167; Chang, B. S. and Hershenson, S. Practical Approachesto Protein Formulation Development. In Rational Design of Stable ProteinFormulations. J. F. Carpenter and M. C. Manning, editors. 2002, PlenumPubl., New York. 1-25; Pharmaceutical Formulation Development ofPeptides and Proteins. 2000, CRC Press, Boca Raton).

As in general, the native (bioactive) structure of proteins is verysensitive towards its surroundings e.g. the composition of theformulation, the container/closure systems, pH and temperature it is adifficult feat to identify a suitable liquid formulation forgonadotropins, different excipients were screened. In the present workthe buffer capacity and real time stability of various hMG formulationsare investigated using an FSH immunoassay, an hCG immunoassay, abioassay, RP-HPLC for determination of oxidized proteins, titration andpH as described below in detail.

2 Product to be Studied

2.1 Materials

2.1.1 Drug Substance (DS)

The hMG-HP drug substance (DS) was manufactured by Instituto MassoneS.A. Argentina hMG-HP DS is received as a powder by FerringPharmaceuticals A/S, Copenhagen, Denmark.

Determination of FSH and LH biological activity in the drug substancewas performed according to the current version of the BritishPharmacopeia (BP). It is also possible, if desired, to carry out thisdetermination according to the USP 35 version. The FSH:LH bioactivity inhMG is 1:1 and therefore the average determined bioactivity of FSH andLH is used for compounding to drug product. Hence, when theconcentration of hMG is given as e.g. 625 IU/mi it equals a bioactivityof 625 IU/mi FSH and 625 IU/mi LH.

2.1.2 Excipients

A list of the excipients used in this work is described in Table 1.

TABLE 1 List of excipients Name Quality Manufacturer Sodium hydroxidepellets Ph Eur, BP, JP, NF Merck Hydrochloric acid, fuming 37% Ph Eur,BP, JP, NF Merck Citric acid Monohydrate Ph Eur, BP, JPE, USP Merckortho-Phosphoric acid 85% Ph Eur, BP, JPE, NF Merck Tri-sodium citratedihydrate Ph Eur, BP, JPE, USP Merck Di-sodium hydrogen phosphate PhEur, BP, JPE, USP Merck dodecahydrate L-histidine EMPROVE ® exp Ph Eur,USD Merck L-methionine USP, multicompendial J. T. Baker L-arginine PhEur, USP Merck L-arginine monohydrochloride Pharma Grade, EP, JP, USPSigma-Aldrich Sucrose EMPROVE ® exp Ph. Eur, BP, JPE, NF MerckD-(+)-trehalose dehydrate ≥99%, cGMP Sigma-Aldrich Mannitol EMPROVE ® PhEur, BP, USP, JPE Merck Lactose monohydrate Ph Eur, BP, NF, JP MerckGlycine EMPROVE ® Ph. Eur., BP, JPE, USP Merck Sodium Chloride EMPROVE,Ph. Eur., BP, USP Merck Polysorbate 20 Ph. Eur, NF, JPE J. T. BakerPoloxamer 188 Suitable for bio. Pharm. Merck Production, Ph. Eur., NFPhenol Ph. Eur, JP, USP Merck Meta-cresol Ph. Eur./USP parenteral gradeHedinger Milli-Q water — Millipore

2.1.3 Container Closure System

For the stability studies, the primary packing materials were glassvials with rubber stoppers and alu/plastic caps, or glass cartridgeswith rubber plungers and crimp caps.

3 Manufacturing Procedure

3.1 Compounding

All formulations are manufactured at lab-scale.

For compounding of drug product solution (DP), stock solutions of eachexcipient and drug substance (DS) are mixed sequentially. Before addinghMG and final dilution to volume, the pH of each formulation isadjusted, when necessary. Stock solutions of all excipients and hMG areprepared in Milli-Q water.

3.2 Sterile Filtration and Aseptic Filling

The formulations for stability are sterile filtered with Millipore PVDF0.22 μm filter. Sterile filtration is performed in a LAF bench usingautoclaved materials.

The filling is performed after filtration. The containers are filledwith sample solution. All vials and cartridges are filled under asepticlike conditions in a LAF bench and immediately closed with rubberstoppers or crimp caps. Outside the LAF bench the filled vials aresealed with aluminium flip-off caps.

4 Storage Conditions

4.1 Storage Conditions

Containers containing drug product formulations are stored ataccelerated conditions for up to 3 months at 30±2° C./65±5% RH and/orfor up to a minimum of 6 months at 25±2° C./60±5% RH. At each storagetemperature, the containers are stored in vertical positions. Cartridgesare stored horizontally. All containers are protected from light.

5 Analytical Methods

The analytical methods used in the studies are described below.

5.1 Titration Procedure

According to USP-NF as described previously, a solution is said to bebuffered if it resists changes in the activity of an ion on the additionof substances that are expected to change the activity of that ion.Buffers are substances or combinations of substances that impart thisresistance to a solution. Buffered solutions are systems in which theion is in equilibrium with substances capable of removing or releasingthe ion. Buffer capacity refers to the amount of material that may beadded to a solution without causing a significant change in ionactivity. It is defined as the ratio of acid or base added (ingram-equivalents/L) to the change in pH units. The capacity of abuffered solution is adjusted to the conditions of use, usually byadjustment of the concentrations of buffer substances.

Buffer capacity is expressed commonly as the number of equivalents (Eq)of strong acid or base that causes one liter of the solution in questionto undergo one unit change in pH giving the unit mEq/(liter×pH) whichwas used for determination of the buffer capacity in the presentinvention. This means that the buffer capacity is defined as number ofmoles (equivalents) of H⁺/OH⁻ needed to change the pH of one literbuffer solution by one unit.

The buffer capacity was determined using placebo (placebo refers here toformulations without active ingredient) formulation adjusted to targetpH as starting point. 0.2 N NaOH/HCl was used to titrate pH up or down.pH was measured after each addition of 0.2 N NaOH/HCl and the volume of0.2 N NaOH/HCl was documented. The amount of 0.2 N NaOH/HCl was plottedas X-values and pH as Y-values. Fitted linear regression was performedaround pH 6.8 for hMG placebo (target pH) and around pH 6.5 forreference placebo (target pH). The buffer capacity can be calculated asμL 0.2 N HCl/NaOH to move the pH by 0.01 pH unit/L DP and asmilliequivalents (mEq) acid or base/(liter×pH unit), as described abovein the definition.

5.2 FSH and hCG Immunoassay

The FSH and hCG immunoassay was determined by sandwich ELFA.

5.3 Oxidized proteins

Oxidization of proteins was determined by RP-HPLC.

5.4 pH

pH was measured according to Ph. Eur.

6 Results

The results of the stability studies, the evaluation of the bufferingagent in a buffer capacity study, as well as the stability results fromthe Design of Experiment (DoE) study are presented below.

TABLE 2 Composition of liquid hMG (600 IU/ml) formulations Batch no.Buffer Surfactant Preservative Stabilizer/Tonicity agent E-01 10 mMPhosphate¹ pH 6.8 0.005 mg/ml polysorbate 20 3.0 mg/ml M-cresol 41.3mg/ml Mannitol E-02 10 mM Phosphate¹ pH 6.8 0.005 mg/ml polysorbate 203.0 mg/ml M-cresol 85.9 mg/ml Trehalose E-03 10 mM Phosphate¹ pH 6.8 0.1mg/ml Poloxamer 188 3.0 mg/ml M-cresol 74.3 mg/ml Sucrose E-04 10 mMPhosphate¹ pH 6.8 0.1 mg/ml Poloxamer 188 3.0 mg/ml M-cresol 18.8 mg/mlGlycine E-05 10 mM Phosphate¹ pH 6.8 0.005 mg/ml polysorbate 20 5.0mg/ml Phenol 67.7 mg/ml Sucrose E-06 10 mM Phosphate¹ pH 6.8 0.005 mg/mlpolysorbate 20 5.0 mg/ml Phenol 6.8 mg/ml NaCl E-07 10 mM Phosphate¹ pH6.8 0.1 mg/ml Poloxamer 188 5.0 mg/ml Phenol 37.6 mg/ml Mannitol E-08 10mM Citrate² pH 6.8 0.005 mg/ml polysorbate 20 3.0 mg/ml M-cresol 71.1mg/ml Sucrose E-09 10 mM Citrate² pH 6.8 0.005 mg/ml polysorbate 20 3.0mg/ml M-cresol 29.3 mg/ml L-arginine HCl E-10 10 mM Citrate² pH 6.8 0.1mg/ml Poloxamer 188 3.0 mg/ml M-cresol 39.5 mg/ml Mannitol E-11 10 mMCitrate² pH 6.8 0.005 mg/ml polysorbate 20 5.0 mg/ml Phenol 74.7 mg/mlTrehalose E-12 10 mM Citrate² pH 6.8 0.1 mg/ml Poloxamer 188 5.0 mg/mlPhenol 64.5 mg/ml Sucrose E-13 10 mM Citrate² pH 6.8 0.1 mg/ml Poloxamer188 5.0 mg/ml Phenol 6.5 mg/ml NaCl E-14 10 mM Histidine pH 6.8 0.005mg/ml polysorbate 20 3.0 mg/ml M-cresol 77.5 mg/ml Sucrose E-15 10 mMHistidine pH 6.8 0.1 mg/ml Poloxamer 188 3.0 mg/ml M-cresol 19.6 mg/mlGlycine ¹10 mM Di-sodium hydrogen phosphate dodecahydrate, pH adjustedwith phosphoric acid ²10 mM Tri-sodium citrate dihydrate, pH adjustedwith citric acid

6.1 Stability of Liquid hMG Formulation/3 Months

Protein molecules can be stabilized by adding excipients to the solutione.g. salts, carbohydrates or amino acids but the degree of stabilizationupon addition of different carbohydrates, salts and amino acids variesmassively between different formulations. Here, the initial stabilitystudies were performed to screen various stabilizers in combination withpreservatives and buffers in a liquid formulation of hMG. Surprisingly,the results show a superior stabilizing effect of L-arginine as shown inTables 3 and 6 for the hCG immunoassay and Tables 4 and 7 for the FSHimmunoassay.

Formulation E-09 exhibits a superior stability compared to all otherformulations. Formulation E-09 is the only formulation which containsL-arginine (see Table 2). Formulation K-01, K-03 and K-04 as well asK-09 exhibit superior stability compared to all other formulations (seeTable 6). These formulations again contain L-arginine (see Table 5). InTable 4, formulation E-09 excels with best stability compared to allother formulations also for the FSH immunoassay and Table 7 confirmsthat formulation K-09 shows the highest FSH immunoassay result comparedto all other formulations.

TABLE 3 hCG immunoassay during 1 month storage at 30 ± 2° C./65 ± 5% RH.Formulation E-09 exhibits superior stability compared to all otherformulations. Formulation E-09 contains L-arginine, see Table 2. hCG [%of initial] 30° C. ± 2° C./65 ± 5% RH Formulation Initial 0.5 month 1month E-01 100 91 85 E-02 100 88 87 E-03 100 87 80 E-04 — — — E-05 10083 81 E-06 100 101 100 E-07 100 89 87 E-08 100 89 81 E-09 100 113 106(L-arginine HCl) E-10 100 95 91 E-11 100 99 89 F-12 100 94 87 E-13 10099 86 E-14 — — — E-15 — — — (Formulation E-04, E-14 and E-15 excludedfrom testing due to coloration)

TABLE 4 FSH immunoassay after 3 months storage at 30 ± 2° C./65 ± 5% RH.Formulation E-09 exhibits the best stability compared to all otherformulations. Formulation E-09 contains L-arginine, see Table 2. FSH [%of initial] 30° C. ± 2° C./65 ± 5% RH Formulation Initial 3 months E-01100 83 E-02 100 77 E-03 100 83 E-04 — — E-05 100 84 E-06 100 86 E-07 10086 E-08 100 84 E-09 100 95 L-arginine E-10 100 83 E-11 100 83 E-12 10089 E-13 100 85 E-14 — — E-15 — — (Formulation E-04, E-14 and E-15excluded from testing due to coloration)

6.2 Stability of Liquid hMG Formulation—3 Months

From Initial Screening Additional Formulations with Arginine wasScreened.

TABLE 5 Composition stability study for liquid hMG 600 IU/ml - Overviewof formulations Batch no. Buffer Surfactant Preservative AntioxidantStabilizer/Tonicity agent K-01¹ 10 mM Citrate⁵ 0.005 mg/ml 3.0 mg/mlM-cresol 1.5 mg/ml 28.0 mg/ml L-arginine HCl pH 6.8 Polysorbate 20L-methionine K-02 1 mM Phosphate⁴ 0.005 mg/ml 3.0 mg/ml M-cresol 1.0mg/ml 21.0 mg/ml Lactose pH 6.8 Polysorbate 20 L-methionine 7.0 mg/mlNaCl K-03 10 mM Citrate⁵ 0.1 mg/ml 5.0 mg/ml Phenol 1.5 mg/ml 25.3 mg/mlL-arginine HCl pH 6.8 Poloxamer 188 L-methionine K-04 10 mM Phosphate⁴0.005 mg/ml 3.0 mg/ml M-cresol 1.5 mg/ml 29.3 mg/ml L-arginine HCl pH6.8 Polysorbate 20 L-methionine K-05² 10 mM Citrate⁵ 0.1 mg/ml 5.0 mg/mlPhenol 1.0 mg/ml 67.4 mg/ml Sucrose pH 6.8 Poloxamer 188 L-methionineK-06 10 mM Citrate⁵ 0.1 mg/ml 3.0 mg/ml M-cresol 1.0 mg/ml 74.6 mg/mlSucrose pH 6.8 Poloxamer 188 L-methionine K-07³ 10 mM Citrate⁵ 0.1 mg/ml3.0 mg/ml M-cresol 1.0 mg/ml 38.3 mg/ml Mannitol pH 6.8 Poloxamer 188L-methionine K-08 10 mM Citrate⁵ 0.1 mg/ml 5.0 mg/ml Phenol 1.0 mg/ml34.7 mg/ml Mannitol pH 6.8 Poloxamer 188 L-methionine K-09⁶ 10 mMCitrate⁵ 0.1 mg/ml 3.0 mg/ml M-cresol 1.5 mg/ml 20 mg/ml L-arginine HClpH 6.8 Poloxamer 188 L-methionine 22.1 mg/ml Sucrose ¹Same asformulation E-09 in table 2 ²Same as formulation E-12 in table 2 ³Sameas formulation E-10 in table 2 ⁴10 mM Di-sodium hydrogen phosphatedodecahydrate, pH adjusted with phosphoric acid ⁵10 mM Tri-sodiumcitrate dihydrate, pH adjusted with citric acid ⁶Strength is 530 IU/ml

TABLE 6 hCG immunoassay during 3 months storage at 25 ± 2° C./60 ± 5%RH. Formulations K-01, K-03, K-04 and K-09 exhibit superior stabilitycompared to all other formulations. Formulation K-01, K-03, K-04 andK-09 contain L-arginine, see Table 5 hCG [% of initial] 25° C.+ 2°C./65 + 5% RH Formulation Initial 1 month 2 months 3 months K-01 100 110105 105 (L-arginine) K-02 100 100 68 57 K-03 100 110 106 104(L-arginine) K-04 100 109 106 113 (L-arginine) K-05 100 94 82 71 K-06100 98 76 80 K-07 100 85 74 67 K-08 100 93 79 71 K-09 100 99 101 100(L-arginine)

Very dearly, the formulations comprising L-arginine are much more stablethan those comprising different stabilizing agents. Compare e.g.formulation K-02 and K-03 after three months.

TABLE 7 FSH immunoassay during 3 months storage at 25 ± 2° C./60 ± 5%RH. Formulation K-09 show highest FSH immunoassay result compared to allother formulations. Formulation K-09 contains L-arginine, see Table 5FSH [% of initial] 25° C.± 2° C./60 ± 5% RH Formulation Initial 1 month2 months 3 months K-01 100 112 112 111 (L-arginine) K-02 100 104 106 87K-03 100 108 120 107 (L-arginine) K-04 100 108 112 112 (L-arginine) K-05100 107 116 111 K-06 100 109 117 115 K-07 100 101 112 104 K-08 100 107107 109 K-09 100 112 120 119 (L-arginine)

The results for hCG are also confirmed for FSH—compare e.g. formulationK-02 and K-03 after three months.

6.3 Buffer Capacity Study

Buffering agents e.g. sodium phosphate and sodium citrate arephysiologically tolerated buffers and are quite commonly added tomaintain the pH in a desired range. Tri-sodium citrate dihydrate andDi-sodium hydrogen phosphate dodecahydrate are evaluated as bufferingagents by means of buffer capacity. The buffer capacity is calculated asthe amount of acid or base needed to move the pH a predefined step, i.e.the amount of μL 0.2 N HCl/NaOH needed to move the pH by 1 pH unit/L DPcalculated as mEq acid or base/(liter×pH unit) as explained above.

TABLE 8 Composition buffer capacity study - Overview Formulations Batchno. Buffer Surfactant Preservative Antioxidant Stabilizer/Tonicity agentC-01 No buffer 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml L- 120 mM L-arginine HCl⁴pH 6.8 Polysorbate 20 Phenol methionine C-02 5 mM Citrate¹ 0.005 mg/ml5.0 mg/ml 1.5 mg/ml L- 120 mM L-arginine HCl⁴ pH 6.8 Polysorbate 20Phenol methionine C-03 10 mM Citrate¹ 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml L-120 mM L-arginine HCl⁴ pH 6.8 Polysorbate 20 Phenol methionine C-04³ 1mM ² Phosphate 0.005 mg/ml 5.0 mg/ml 1.0 mg/ml L- 31.76 mg/ml Na₂SO₄ ×10 H₂O pH 6.5 Polysorbate 20 Phenol methionine (14 mg/ml Sodiumsulphate) ¹Tri-sodium citrate dihydrate ² 0.8 mM Na₂HPO₄ × 12 H₂O andapprox. 0.2 mM H₃PO₄ to pH 6.5 ³Reference placebo ⁴Equal to 25.3 mg/mlL-arginine HCl

The result of the titration conducted to determine buffer capacity isdepicted in FIG. 1 . The slope of the curves shows the buffer capacity,e.g. a steep slope represents a low buffer capacity. From FIG. 1 it canbe observed that batch C-04 (reference formulation) has the lowestbuffer capacity. In the relevant pH area 6.8±0.3 for hMG, all hMGformulations C-01 (no buffer), C-02 (5 mM Tri-sodium citrate dihydrate)and C-03 (10 mM Tri-sodium citrate dihydrate) have similar buffercapacity and a higher buffer capacity than the reference formulationcontaining 1 mM Di-sodium hydrogen phosphate dodecahydrate. The targetpH for the reference formulation is 6.5. Formulation C-01, C-02 and -03are compared with the reference which is known to have a stable pH.Numerous stability studies of the reference formulation C-04 have beenconducted without any observed drifts in pH.

The following Table 9 shows the results of the buffer capacity study:

TABLE 9 Buffer capacity study. In the formulation relevant pH area 6.8for hMG, all hMG formulations have a higher buffer capacity than thereference placebo formulation. Buffer capacity Buffer expressed as:capacity mEq acid or around a α base/(liter × Batch Buffer target pH pHrange (slope) pH unit) C-01 No buffer 6.8 6.564-6.947 0.001507 1.32 C-01No buffer 7.0 6.786-7.193 0.000822 2.44 C-02 5 mM Tri-sodium 6.86.599-6.951 0.000672 2.96 citrate dihydrate C-03 10 mM Tri-sodium 6.86.572-6.946 0.000750 2.66 citrate dihydrate C-04 1 mM Di-sodium 6.56.199-6.752 0.002700 0.74 (reference) hydrogen phosphate dodecahydrateC-04 1 mM Di-sodium 6.8 6.582-6.982 0.002091 0.96 (reference) hydrogenphosphate dodecahydrate (C-01 and C-04 is mentioned twice because thebuffer capacity was calculated in pH ranges around two different pHvalues respectively)

Surprisingly, the buffer capacity of the hMG formulation with no buffer(C-01) is higher than the reference formulation C-04. The main excipientcomponent in the hMG formulation is L-arginine at a concentration of 120mM. The pKa values for L-arginine are pKa₁=2.17; pKa₂=9.04 and pKa₃=12.5(Handbook of Pharmaceutical Excipients. 2015, Pharmaceutical Press,London). It is very unexpected that arginine exhibits sufficientbuffering behaviour far from the pKa values.

6.4 Oxidation Studies

After 3 months of storage at 30±2° C./65±5% RH, formulation E-09containing L-arginine (see Table 5) has increased the level of oxidizedproteins by 241% of the initial value (see table 10 below). This is avery high amount and there are instances where such high amounts ofoxidation are not desirable.

Therefore, further studies were conducted to research whether thisproblem could be overcome.

There are several choices of antioxidants that can be used in proteinformulations. Methionine can be added to prevent oxidation, by aproposed mechanism of competing with oxidation of the methionineresidues in the proteins. The result of adding methionine to aformulation containing arginine is shown in Table 10. Formulation K-01,K-03, K-04 and K-09 contain arginine and methionine and the level ofoxidized proteins is clearly and advantageously reduced. FormulationK-01 (with methionine) is the same formulation as formulation E-09(without methionine). Comparing these two batches it can be seen thatthe level of oxidized proteins is reduced tremendously by methionine.

TABLE 10 Oxidized proteins % increase from initial during 3 monthsstorage at 30 ± 2° C./65 ± 5% RH. Formulation E-09 contains arginine andno methionine. Formulation K- 01, K-03, K-04 and K-09 contain arginineand methionine, see Table 5 Oxidized proteins [% increase from initial]30° C. ± 2° C./65 ± 5% RH Formulation 3 months E-01 44 E-02 39 E-03 47E-04 — E-05 37 E-06 43 E-07 36 E-08 51 E-09 241 (arginine and nomethionine) E-10 55 E-11 47 E-12 53 E-13 46 E-14 — E-15 — K-01 24(arginine and methionine) K-03 12 (arginine and methionine) K-04 8(arginine and methionine) K-09 13 (arginine and methionine) (FormulationE-04, E-14 and E-15 excluded due to coloration)

In conclusion, these two studies demonstrate a tremendously stabilizingeffect of arginine and an anti-oxidizing effect even of low amounts ofmethionine in a formulation of gonadotropins.

6.5 DoE Stability Study

Combining the results from the above studies, and the buffer capacitystudy, a DoE study was performed to investigate the interactions betweenarginine and methionine and to determine the optimal concentrations ofthese two excipients.

TABLE 11 Compositions for DoE stability study for liquid hMG 600 IU/ml-overview of formulations Batch Stabilizer/Isotonicity no. Buffer²Surfactant Preservative Antioxidant agent D-01 No buffer 0.005 mg/ml 5.0mg/ml 1.5 mg/ml L- 80 mM pH 6.8 Polysorbate 20 Phenol methionineL-arginine HCl D-02 No buffer 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml L- 160 mMpH 6.8 Polysorbate 20 Phenol methionine L-arginine HCl D-03 No buffer0.005 mg/ml 5.0 mg/ml 0.8 mg/ml L- 120 mM pH 6.8 Polysorbate 20 Phenolmethionine L-arginine HCl D-04 No buffer 0.005 mg/ml 5.0 mg/ml 0.8 mg/mlL- 120 mM pH 6.8 Polysorbate 20 Phenol methionine L-arginine HCl− D-05No buffer 0.005 mg/ml 5.0 mg/ml 0.1 mg/ml L- 160 mM pH 6.8 Polysorbate20 Phenol methionine L-arginine HCl D-06 No buffer 0.005 mg/ml 5.0 mg/ml0.1 mg/ml L- 80 mM pH 6.8 Polysorbate 20 Phenol methionine L-arginineHCl D-07³ No buffer 0.1 mg/ml 5.0 mg/ml 1.5 mg/L- 120 mM pH 6.8Poloxamer 188 Phenol methionine L-arginine HCl D-08³ 10 mM Citrate¹ 0.1mg/ml 5.0 mg/ml 1.5 mg/L- 120 mM pH 6.8 Poloxamer 188 Phenol MethionineL-arginine HCl ¹Tri-sodium citrate dehydrate ²pH is adjusted with 0.2NHCl/NaOH for formulations without buffer and 0.2N NaOH/0.5M ³Citric acidMonohydrate for formulation with Tri-sodium citrate dihydrate bufferIncluded as reference formulations

6.5.1 FSH Immunoassay

Stability results of FSH immunoassay during storage for 3 months at 25 12° C./601 5% RH are listed in Table 12.

TABLE 12 Results of FSH immunoassay during storage at 25 ± 2° C./60 ± 5%RH. The full description of all formulations is listed in Table 11. FSH[IU/ml] 25° C. ± 2° C./60 ± 5% RH Formulation Initial 1 month 2 months 3months D-01 516 501 468 462 D-02 521 492 503 478 D-03 511 503 513 474D-04 515 505 490 477 D-05 532 521 513 473 D-06 518 488 458 493 D-07 518528 482 490 D-08 514 499 472 481

Statistical calculation was performed for FSH activity [U/ml] toevaluate the influence and interaction of arginine and -methionine. Theresult of the statistical evaluation confirms that L-arginine has astatistical significant influence on FSH immunoassay stability results.Methionine has no statistical significant influence on FSH immunoassaystability results. There is no statistical significant interactionbetween arginine and methionine for this response parameter.

6.5.2 hCG Immunoassay

Stability results of hCG immunoassay during storage for 3 months at25±2° C./60±5% RH are listed in Table 13 Table.

TABLE 13 Results of hCG immunoassay during storage at 25 ± 2° C./60 ± 5%RH. The full description of all formulations is listed in Table 11. hCG[IU/ml] 25° C. ± 2° C./60 ± 5% RH Formulation Initial 1 month 2 months 3months D-01 83 82 75 56 D-02 87 83 93 76 D-03 93 93 88 70 D-04 86 91 8770 D-05 90 98 92 85 D-06 87 85 72 61 D-07 93 92 88 71 D-08 91 96 94 81

Statistical calculation was performed for hCG activity [IU/ml] toevaluate the influence and interaction of arginine and methionine. Theresult of the statistical evaluation confirms that arginine has astatistical significant influence on hCG immunoassay stability results.Methionine content has a minor influence at 25° C. on hCG immunoassaystability results. It becomes clear that over a low (0.1 mg/ml)concentration of methionine no further increase in stability byincreasing the concentration even up to 1.5 mg/ml is detectable. Thereis no statistical significant interaction between arginine andmethionine for this response parameter.

6.5.3 Oxidized Proteins

In the above stability studies it was surprisingly observed that thepresence of arginine has a tremendously stabilizing effect. However,addition of arginine resulted in an increased level of oxidizedproteins. Methionine can be added to prevent this oxidation and thisstudy investigates the concentration balance between arginine asstabilizer and methionine as antioxidant.

Stability results of amount of oxidized proteins [% increase frominitial] during storage for 6 months at 25±2° C./60±5% RH and 3 month at30±2° C./65±5% RH for formulations with arginine and Methionine (in theconcentration range 0.1-1.5 mg/ml) are listed below. For comparisonformulation E-09 with arginine and without methionine is also includedin Table 14 Table.

TABLE 14 Results of oxidized proteins during storage at 25 ± 2° C./60 ±5% RH and 30° C. ± 2° C./65 ± 5% RH. The full description of allformulations is given in Table 11. Oxidized proteins [% increase frominitial] 30° C. ± 2° C./65 ± 5% RH 25° C. ± 2° C./60 ± 5% RH Formulation1 month 2 months 3 months 1 month 2 months 3 months 6 months D-01 5.54.1 8.3 10.1 4.1 9.6 6.9 D-02 9.9 8.5 10.3 11.7 11.7 6.6 8.5 D-03 10.910.0 11.4 12.3 13.3 11.8 12.8 D-04 8.9 8.4 8.9 8.9 11.2 9.8 8.9 D-0512.9 13.8 13.8 13.3 20.5 11.0 15.2 D-06 7.0 9.8 11.6 10.7 7.9 9.3 11.2D-07 14.0 10.1 11.1 14.5 13.5 15.0 10.1 D-08 7.6 9.5 7.6 13.3 9.0 11.87.6 E-09 (no L-methionine) — — 241 — — — —

From Table 14 Table it is surprisingly seen that even small quantitiesof methionine are enough to prevent oxidation in the entireconcentration range of arginine.

6.5.4 Summary of DoE

To conclude and summarize the results of the DoE, the response optimizerin Minitab was applied, see FIG. 2 . The Minitab Response Optimizer toolshows how different experimental factor settings e.g. arginine andmethionine concentrations affect the predicted responses e.g. the FSHimmunoassay [IU/ml] and the hCG immunoassay [IU/ml] for the factorialdesign. The optimization plot shows the effect of each factor (columns)on the responses (rows). The vertical bold lines on the graph representthe current factor settings. The numbers displayed at the top of acolumn show the current factor level settings (in squared brackets). Thehorizontal dashed lines and corresponding numbers represent theresponses for the current factor level. The response optimizer will bebased on 25° C. results. The result of applying the response optimizeris depicted FIG. 2 . The best stability (composite desirability) isobtained at high concentrations of arginine and low concentrations ofmethionine.

6.6 pH Study

In the buffer capacity study it was shown that a buffering agent was notnecessary in the hMG formulation, by using only arginine to stabilizethe pH in the desired pH range.

To confirm that the pH is maintained during storage, it was measuredduring 6 months of storage at 25±2° C./60±5% RH and 3 months at 30±2°C./65±5% RH. The results are listed in Table 15.

Target pH for all formulations was pH 6.8 and pH of all formulations was6.8 at the initial time point. pH was fairly stable but increasedslightly to about 6.9 after 2 months and maintained a pH around 6.9 upto 6 months of storage. Results up to 6 months of storage confirmstability of pH in the tested concentration range of arginine.

TABLE 15 Results of pH study 30° C. ± 2° C./ 25° C. ± 2° C./ 65 ± 5% RH60 ± 5% RH pH 2 3 2 3 6 Formulation Initial month months months monthsmonths D-01 6.8 6.9 6.9 6.9 6.9 6.9 D-02 6.8 6.9 6.9 6.9 6.9 6.8 D-036.8 6.9 6.9 6.9 6.9 6.8 D-04 6.8 6.9 6.9 6.9 6.9 6.8 D-05 6.8 6.9 6.96.9 6.9 6.8 D-06 6.8 6.9 6.9 6.9 6.9 6.8 D-07 6.8 6.9 6.9 6.9 6.9 6.8D-08 6.8 6.9 6.9 6.9 6.9 6.9

7 Conclusion

Very surprisingly, the buffer capacity of the hMG formulation with nobuffer is higher than the reference with 1 mM Di-sodium hydrogenphosphate dodecahydrate. The main excipient component in the hMGformulation is arginine. The pKa values for arginine are pKa₁=2.17;pKa₂=9.04 and pKa₃=12.5. It is unexpected that arginine exhibitssufficient buffering behaviour far from its pKa values.

The results however clearly show that no-additional buffering agent isneeded in the hMG formulation in the current pH range. Arginine alone issufficient to maintain the pH in the desired pH range. Stability of thepH was confirmed in the DoE study testing a concentration range ofarginine during 6 months storage at 251 2° C./6015% RH and 3 monthsstorage at 301 2° C./65±5% RH which confirmed the observations in thebuffer capacity study.

Addition of arginine to the inventive liquid hMG formulation was shownto have a tremendously stabilizing effect on liquid hMG formulations.However, arginine was also found to result in a high level of oxidizedproteins. Addition of methionine was shown to prevent oxidation comparedto a formulation without methionine.

During 6 months of storage at 25±2° C./60±5% RH and 3 months of storageat 30±2° C./65±5% RH, it was shown that even a small concentration ofmethionine prevents oxidation.

Concluding the results from these studies it is clearly seen that theaddition of arginine is sufficient to maintain pH at the desired pHlevel. Addition of arginine stabilizes the tested gonadotropinformulation tremendously in a liquid formulation. No other of the testedamino acid, sugar or salt showed a similar stabilizing effect. The levelof oxidized proteins is increased significantly by addition of arginine,however addition of even low amounts of methionine prevents proteinoxidation. Surprisingly, even small quantities of methionine aresufficient to prevent oxidation independently of the concentration ofarginine.

Example 2

The present inventors further confirmed that the present advantageousinventive formulation would also be suitable to stabilize the respectiverecombinant gonadotropin formulation.

To that avail, recombinant FSH and recombinant hCG (with the sequencesas described above, respectively) were prepared according to well knownmethods.

An accelerated stability study was carried out for two formulations,comprising rhCG or rFSH, respectively, as described below.

The observed surprising stabilising effect of arginine has also beenconfirmed in recombinant proteins. Both rFSH and rhCG have beenformulated in 5 mg/ml phenol, 0.15 mg/ml L-methionine, 150 mM arginineHCl, 0.005 mg/ml polysorbate 20, pH 6.8. To simplify the analysis of theprotein stability, rFSH and rhCG were formulated in differentcontainers.

TABLE 16 Immunoactivity (IU/ml] 1 month, 2 months, 4 months, 16 months,Initial 30° C. 30° C. 30° C. 5° C. FSH [33.3 μg/ml] 464 444 456 469 435rhCG [50 μg/ml] 711 680 656 600 685 SEC (Purity of rFSH, %) 1 month, 2months, 4 months, 16 months, Initial 30° C. 30° C. 30° C. 5° C. FSH[33.3 μg/ml] 98.9 98.6 98.3 97.9 96.9 HIC (Purity of rhCG, %) 1 month, 2months, 4 months, 14 months, Initial 30° C. 30° C. 30° C. 5° C. rhCG [50μg/ml] 98.8 96.8 93.3 89.4 99.9

This fully confirms that the present inventive formulation witharginine, but no additional buffer, and with a low amount of methionineis suitable to stabilized recombinant gonadotropins as well.

Example 3

Further studies were conducted, wherein the following composition wasassessed:

TABLE 17 Composition for liquid hMG 625 IU/ml formulation BatchStabilizer/Tonicity no. Buffer¹ Surfactant Preservative Antioxidantagent J-01 No buffer 0.005 mg/ml 5.0 mg/ml 0.15 mg/ml 150 mM pH 6.8Polysorbate 20 Phenol L-Methionine L-arginine HCl ¹pH is adjusted with0.2N HCl/NaOH

Stability of Target Formulation

Combining all previous results, the good stability was confirmed for theabove formulation, as follows:

TABLE 18 FSH immunoassay Results of FSH immunoassay during storage at25° C. ± 2° C./60 ± 5% RH, and 12 months at 5 ± 3° C. 25° C. ± 2° C./ 60± 5% RH 5° C. ± 3° C. FSH [% of initial] 1 2 3 3 12 Formulation Initialmonth months months months months J-01 100 95.2 95.9 98.8 96.8 100.5 Theresults confirm the stability as determined by FSH immunoassay

Stability results of FSH immunoassay during storage for 3 months at 252° C./60±5% relative humidity (RH) and 12 months at 5±3° C.:

The results confirm the stability as determined by FSH immunoassay.

TABLE 19 hCG immunoassay Results of hCG immunoassay during storage at 25± 2° C./60 ± 5% RH and 12 months at 5 ± 3° C. 25° C. ± 2° hCG [% ofinitial] C./60 ± 5% RH 5° C. ± 3° C. Formulation Initial 1 month 3months 12 months J-01 100 88.3 94.1 89.5

Stability results of hCG immunoassay during storage for 1 month at 25±2°C./60±5% RH is listed in Table 19 and 12 months at 5±3° C.

The results confirm the stability as determined by hCG immunoassay.

TABLE 20 Oxidized proteins Results of oxidized proteins during storageat 25 ± 2° C./60 ± 5% RH and 12 months at 5 ± 3° C. Oxidized proteins [%increase from initial] 25° C. ± 2° C./60 ± 5% RH 5° C. ± 3° C.Formulation 1 month 2 months 3 months 3 months 12 months J-01 1.8 5.77.9 2.6 11

Stability results of amount of oxidized proteins [% increase frominitial] during storage for 3 months at 25±2° C./60±5% RH and 12 monthsat 5±3° C.

pH

Stability results of pH during storage for 3 months at 25±2° C./60±5% RHand 12 months at 5±3° C. show a stable pH.

TABLE 21 Results of pH during storage at 25° C. ± 2° C./ 60 ± 5% RH and12 months at 5 ± 3° C. 25° C. ± 2° C./ 60 ± 5% RH 5° C. ± 3° C. pH 1 2 33 12 Formulation Initial months months months months months J-01 6.9 6.76.8 6.7 16.7 6.8

Example 4

In addition to the above, the present inventors also performed abioassay study (6 months storage at 25° C. for the DoE batches).

FSH Bioassay (Steelman-Pohley)

Stability results of the FSH bioassay during storage for 6 months at25±2° C./60±5% RH are listed in Table 22.

TABLE 22 Results of FSH bioassay during storage at 25 ± 2° C./60 ± 5%RH. The full description of all formulations is given in Table 11. FSH[% of stated potency (600 IU/ml)] 25° C. ± 2° C./60 ± 5% RH Formulation6 months D-01 98.8 D-02 100.7 D-03 102.0 D-04 104.7 D-05 103.4 D-06101.0 D-07 102.6 D-08 104.9

The results confirm the stability, as determined by FSH bioassay.

LH Bioassay (Seminal Weight Gain)

Stability results of LH bioassay during storage for 6 months at 25±2°C./60±5% RH is listed in Table 23.

TABLE 23 Results of LH bioassay during storage at 25 ± 2° C./60 ± 5% RH.The full description of all formulations is given in Table 11. LH [% ofstated potency (600 IU/ml)] 25° C. ± 2° C./60 ± 5% RH Formulation 6months D-01 87.8 D-02 101.3 D-03 90.9 D-04 95.6 D-05 97.5 D-06 95.0 D-0797.3 D-08 100.1

The results confirm the stability, as determined by LH bioassay.

Furthermore, the composition of Example 3 was also tested in an FSH andLH bioassay, with the following results:

Stability results of FSH bioassay during storage for 3 months at 25±2°C./60±5% RH and 12 months at 5±3° C. are listed in Table 24:

TABLE 24 Results of FSH bioassay during storage at 25° C. ± 2° C./ 60 ±5% RH and 12 months at 5 ± 3° C. FSH [% of stated 25° C. ± 2° C./potency 60 ± 5% RH 5° C. ± 3° C. (625 IU/ml)] 1 2 3 3 12 FormulationInitial month months months months months J-01 94.7 93.6 94.5 97.6 95.896.1

The results confirm the stability determined by FSH bioassay.

Stability results of LH bioassay during storage for 3 months at 25±2°C./60±5% RH and 12 months at 5±3° C. is listed in Table 25.

TABLE 25 Results of LH bioassay during storage at 25° C. ± 2° C./ 60 ±5% RH and 12 months at 5 ± 3° C. LH [% of stated 25° C. ± 2° C./ potency60 ± 5% RH 5º C. ± 3° C. (625 IU/ml)] 1 2 3 3 12 Formulation Initialmonth months months months months J-01 102.1 102.2 99.0 100.0 101.5100.0

The results confirm the stability determined by LH bioassay.

8 Abbreviations and Definitions

-   -   BP British Pharmacopeia    -   DoE Design of experiment    -   DS Drug substance    -   FSH Follicle-stimulating hormone    -   hCG Human chorionic gonadotropin    -   hMG Human menopausal gonadotropin    -   hMG-HP Human menopausal gonadotropin-Highly purified    -   JP Japanese Pharmacopeia    -   LH Luteinizing hormone    -   MD Multiple dose    -   NF National Formulary    -   Ph Eur European Pharmacopeia    -   PS 20 Polysorbate 20    -   USP U.S. Pharmacopeia    -   WFI Water for injection

1-15. (canceled)
 16. A liquid pharmaceutical formulation, comprising agonadotropin, and arginine in an amount of 50 to 160 mM, wherein theformulation does not additionally comprise a buffer, and wherein the pHof the formulation is between 6.0 and 7.5.
 17. The pharmaceuticalformulation of claim 16, wherein the gonadotropin comprises hCG (humanchorionic gonadotropin), and optionally FSH and/or LH.
 18. Thepharmaceutical formulation of claim 16, wherein the gonadotropincomprises hMG (human menopausal gonadotropin).
 19. The pharmaceuticalformulation of claim 16, wherein the gonadotropin comprises humanorigin, urinary-derived FSH, LH and/or hCG.
 20. The pharmaceuticalformulation of claim 16, wherein the gonadotropin comprises recombinantFSH, LH and/or hCG.
 21. The pharmaceutical formulation of claim 16,further comprising a preservative.
 22. The pharmaceutical formulation ofclaim 21, wherein the preservative is phenol.
 23. The pharmaceuticalformulation of claim 22, wherein the phenol is present in an amount of4-6 mg/mL.
 24. The pharmaceutical formulation of claim 22, wherein thephenol is present in an amount of 5 mg/mL.
 25. The pharmaceuticalformulation of claim 16, further comprising a surface-active agent. 26.The pharmaceutical formulation of claim 25, wherein the surface-activeagent is a polysorbate.
 27. The pharmaceutical formulation of claim 25,wherein the surface-active agent is polysorbate
 20. 28. Thepharmaceutical formulation of claim 27, wherein the polysorbate 20 ispresent in an amount of 0.001-0.05 mg/mL.
 29. The pharmaceuticalformulation of claim 27, wherein the polysorbate 20 is present in anamount of 0.005 mg/mL.
 30. The pharmaceutical formulation of claim 16,wherein the arginine is L-arginine HCl.
 31. The pharmaceuticalformulation of claim 18, wherein the hMG is present in an amount of300-900 IU/mL.
 32. The pharmaceutical formulation of claim 18, whereinthe hMG is present in an amount of 500-700 IU/mL.
 33. A method oftreating infertility in a subject in need thereof, the method comprisingadministering the pharmaceutical formulation of claim 16 to the subject.34. The method of claim 33, wherein the treating is a treatment ofovulation induction (OI), assisted reproductive techniques (ART), and/orhypogonadotrophic hypogonadism in men.
 35. A method for stabilization ofa liquid pharmaceutical formulation comprising hMG, the methodcomprising: providing a sample of urine from a human woman; extractinghMG from the sample; compounding the extract with arginine; optionallyadding phenol and polysorbate; and adjusting the pH of the formulationto between 6.0 and 7.5; wherein the formulation does not additionallycomprise a buffer.